(1) Field of the Invention
The present invention generally relates to assays for enzyme activity and for enzyme inhibitors. More specifically, the invention relates to assays for detecting methionine aminopeptidases and inhibitors of methionine aminopeptidases.
(2) Description of the Related Art
In all living cells, protein synthesis is initiated with an AUG codon, specifying methionine as the N-terminal amino acid in nascent proteins. In both prokaryotes and eukaryotes, this N-terminal methionine will be removed by a methionine aminopeptidase (MAP) (EC 3.4.11.18) if the penultimate amino acid residue is small and uncharged, e.g., Gly, Ala, Ser, Cys, Thr, Pro, and Val, although methionine cleavage activity by MAP is reduced when the N-terminal three amino acids are Met-Thr-Pro or Met-Val-Pro (Moerschell et al., 1990, J. Biol. Chem. 265, 19638-19643; Tsunasawa et al., 1985, J. Biol. Chem. 260, 5382-5391). Removal of the N-terminal methionine is essential for certain proteins to function normally in vivo. For example, the removal of the initiator methionine is often required for subsequent N-terminal modifications, such as N-myristoylation, which is essential for the normal function of various signal transduction proteins, cancer cells, protein targeting moieties, and enzymes (Gordon et al., 1991, J. Biol. Chem. 266, 8647-8650; Duronio et al., 1989, Science 243, 796-800).
Methionine aminopeptidases have been isolated and cloned from several organisms, including E. coli and several other eubacteria, yeast, rat, and various archaea. Currently discovered MAPs have been categorized into two types, type 1 MAP and type 2 MAP, based on structural and sequence similarities. Eubacteria have type 1, archaea have type 2, and eukaryotes have both types. In eukaryotes, null mutants in either type are viable but slow growing, but null mutants of both MAP types are nonviable (Li and Chang, 1995, Proc. Natl. Acad. Sci. USA 92, 12357-12361; Li and Chang, 1996, Biochem. Biophys. Res. Commun. 227, 152-159; Bradshaw et al., 1998, Trends Biochem. Sci. 21, 285-286). Similarly, knockouts of the bacterial MAP1 gene are lethal (Ben-Bassat et al., 1987, H. Bacteriol. 169, 751-757). Thus, MAP activity is essential for normal functioning of prokaryotic and eukaryotic cells.
Aside from their role in cleaving the initiator methionine of proteins, MAPs affect other cellular functions. For example, human type 2 MAP also serves as eukaryotic initiation factor-2, which regulates protein synthesis (U.S. Pat. No. 5,885,820). Also, the mode of action of fumagillin-type angiogenesis inhibitors is the irreversible inhibition of type 2 MAP (Griffith et al., 1997, Chem. Biol. 4, 461-471; Liu et al., 1998, Science 282, 1324-1327; Lowther et al., Proc. Natl. Acad. Sci. USA 95, 12153-12157; Sin et al., 1997, Proc. Natl. Acad. Sci. USA 94, 6099-6103) thus indicating an essential role of type 2 MAP in angiogenesis.
Because of the crucial role of MAPs in prokaryotic and eukaryotic functions, there is an interest in the discovery of additional inhibitors of these enzymes, which may serve as antibiotics or as chemotheraputic agents which inhibit angiogenesis in tumors. However, current methods for monitoring MAP activity are inadequate for this task. These methods include the colorimetric ninhydrin method (Doi et al., 1981, Anal. Biochem. 118, 173-184); amino acid oxidase treatment followed by peroxidase reaction and o-dianisidine color development (Carter and Miller, 1984, J. Bacteriol. 159, 453-459); amino acid analysis via ion exchange chromatography followed by postcolumn derivatization with ninhydrin (Moore et al., 1958, Anal. Biochem. 30, 1185-1190), fluorescamine (Stein et al., 1973, Arch. Biochem. Biophys. 155, 202-212), or o-phthalaldehyde/.beta.-mermercaptoethanol (Roth, 1971, Anal. Chem 43, 880-882); precolumn derivatization of amino acids followed by reverse phase HPLC chromatography (Cohen and Strydom, 1988, Anal. Biochem. 174, 1-16; Zuo et al., 1994, Anal. Biochem. 222, 514-516); and separation of substrate peptides and products by reverse phase HPLC with on-line UV detection of each separated compound (Larrabee et al., 1999, Anal. Biochem. 269, 194-198; Walker et al., 1999, Biotechnol. Appl. Biochem. 29, 157-163). These methods are either not sensitive or accurate enough for quantitative assays, or are not rapid enough for high-throughput screening procedures. Thus, there is a need for new assays for MAP activity which are rapid and quantitative enough for use in procedures requiring high-throughput MAP analysis, such as screening for MAP inhibitors.